Osteoporosis is the most common bone disease in humans. Primary osteoporosis, accompanied by increased susceptibility to fractures, results from a progressive reduction in skeletal bone mass. It is estimated to affect 15-20 million individuals in the USA alone. Its basis is an age-dependant imbalance in bone remodeling, i.e., in the rates of formation and resorption of bone tissue.
In the USA about 1.2 million osteoporosis-related fractures occur in the elderly each year including about 538,000 compression fractures of the spine, about 227,000 hip fractures and a substantial number of early fractured peripheral bones. Between 12 and 20% of the hip fractures are fatal because they cause severe trauma and bleeding, and half of the surviving patients require nursing home care. Total costs from osteoporosis-related injuries now amount to at least $10 billion annually in the USA (Riggs, New England Journal of Medicine, 327:620-627 (1992)).
Osteoporosis is most common in postmenopausal women who, on average, lose 15% of their bone mass in the 10 years after menopause. This disease also occurs in men as they get older and in young amenorrheic women athletes. Despite the major, and growing, social and economic consequences of osteoporosis, the availability of reliable assays for measuring bone resorption rates in patients or in healthy subjects is very limited. Other disorders entailing (and correlated with) abnormalities in collagen metabolism include Paget's disease, Marfan's syndrome, osteogenesis imperfecta, neoplastic growth in collagenous tissue, dwarfism, rheumatoid arthritis, osteo-arthritis and vasculitis syndrome.
Three known classes of human collagen have been described to date. The Class I collagens, subdivided into type I, II, III, V, and XI are known to form fibrils. The amino-acid sequence of type I-III (to the extent it has been elucidated) is given in Appendix A of WO95/08115.
Collagen type I accounts for more than 90% of the organic matrix of bone. Therefore, in principle, it is possible to estimate the rate of bone resorption by monitoring the degradation of collagen type I. Likewise, a number of other disease states involving connective tissue can be monitored by determining the degradation of collagen. Examples are collagen type II degradation associated with rheumatoid arthritis and osteo-arthritis and collagen type III degradation in vasculitis syndrome.
Amino acid sequences of human type III collagen, human pro .alpha.1 (II) collagen, and the entire prepro .alpha.1 (III) chain of human type III collagen and corresponding cDNA clones have been investigated and determined by several groups of researchers; see Loil et al., Nucleic Acid Research 12:9383-9394 (1984): Sangiorgi et al., Nucleic Acids Research, 13:2207-2225 (1985); Baldwin et al., Biochem J., 262:521-528 (1989); and Ala-Kokko et al., Biochem. J., 260:509-516 (1989).
Type I, II and III collagens are all formed in the organism as procollagen molecules, comprising N-terminal and C-terminal propeptide sequences, which are attached to the core collagen molecules. After removal of the propeptides, which occurs naturally in vivo during collagen synthesis, the remaining core of the collagen molecule consists largely of a triple-helical domain having terminal telopeptide sequences (one N-terminal, one C-terminal) which are non-triple-helical. These telopeptide sequences have an important function as sites of intermolecular crosslinking of collagen fibrils extracellularly. The alphahelical region also includes crosslinkable sites.
Intermolecular cross-links provide collage fibrils with biomechanical stability. The formation of these cross-links is initiated by modification of lysine and hydroxylysine residues to the corresponding aldehydes. Several of these residues located on adjacent chains of collagen will spontaneously form different intermolecular cross-links. The exact position of the sites for cross-linking on collagen telopeptides and from the helical region has been previously described. See, for example, Kuhn, K., in Immunochemistry of the Extracellular Matrix, Furthmayr, H., ed. 1:1-29 (1982). Two are aldehyde sites, one in each telopeptide region. The other two sites are hydroxylysine symmetrically placed at about 90 residues from each end of the molecule. When collagen molecules pack into fibrils, these latter sites in the helical region align and react with telopeptide aldehydes in adjacent molecules.
As illustrated by formula in EP-0394296 discussed below, the two 3-hydroxypyridinium cross-links have been found to be hydroxylysyl pyridinoline (also known as "pyridinoline") and lysyl pyridinoline (also known as "deoxypyridinoline"). These cross-linking compounds are naturally fluorescent. Some hydroxylysyl pyridinoline cross-links are found to be glycosylated as discussed for instance EP-A-0424428.
However, as described in Last et al., Int. J. Biochem. 22:559-564 (1990), other crosslinks occur naturally in collagen.
A number of known assays are directed at measuring the amount of 3-hydroxypyridinium or other crosslinks. See, for background and as examples, Wu and Eyre, Biochemistry, 23:1850 (1984); Black et al., Annals of the Rheumatic Diseases, 45:969-973 (1986); and Seibel et al., The Journal of Dermatology, 16:964 (1989). These reports describe hydrolyzing peptides from body fluids and then looking for the presence of free 3-hydroxypyridinium residues.
Assays for determination of the degradation of type I, II, and III collagen are disclosed in EP-0394296 and U.S. Pat. Nos. 4,973,666 and 5,140,103. However, these patents only describe non-isomerized collagen fragments containing the cross-linker 3-hydroxypyridinium. Furthermore, the above mentioned assays require tedious and complicated purifications from urine of collagen fragments containing 3-hydroxypyridinium to be used for the production of antibodies and for antigens in the assays.
At present very few clinical data using the approach described in U.S. Pat. Nos. 4,973,666 and 5,140,103 are available. Particularly, no data concerning the correlation between the urinary concentration (as determined by methods described in the above mentioned patents) of 3-hydroxypyridinium containing C-terminal telopeptides of type I collagen and the actual bone loss (as determined by repeated measurements by bone densitometry) have been published. The presence of 3-hydroxypyridinium containing telopeptides in urine requires the proper formation in bone tissue of this specific cross-linking structure at various times before the bone resorbing process. Very little information on these processes is available and it would be desirable to avoid this dependence of the correct formation of the cross-linking structure.
GB Patent Application No. 2205643 reports that the degradation of type III collagen in the body can be quantitatively determined by measuring the concentration of an N-terminal telopeptide from type III collagen in a body fluid. This method uses antibodies generated to N-terminal telopeptides released by bacterial collagenase degradation of type III collagen, said telopeptides being labelled and used in the assay.
The development of a monoclonal antibody raised against pepsin-solubilized type I collagen is described in Werkmeister et al., Eur. J. Biochem. 1987:439-443 (1990). The antibody is used for immunohistochemical staining of tissue segments and for measuring the collagen content in cell cultures. The measurements are not carried out on body fluids.
EP Patent Application No. 0505210 describes the development of antibody reagents by immunization with purified cross-linked C-terminal telopeptides from type I collagen. The immunogen is prepared by solubilizing human bone collagen with bacterial collagenase. The antibodies thus prepared are able to react with both cross-linked and non-cross-linked telopeptides, and with cross-linkers other than pyridinoline.
WO94/03813 describes a competitive immunoassay for detecting collagen or collagen fragments in a sample wherein a binding partner containing a synthetic linear peptide corresponding to the non-helical C-terminal or N-terminal domain of collagen is incubated with an antibody to the linear synthetic peptide and the sample, and wherein the binding of the antibody to the binding partner is determined.
WO95/08115 relates to assay methods in which collagen fragments in a body fluid are determined by reaction with an antibody which is reactive with a synthetic peptide. The assay may be a competition assay in which the sample and such a peptide compete for an antibody, possibly a polyclonal antibody raised against fragments of collagen obtained by collagenase degradation of collagen. Alternatively, it may be an assay in which an antibody, possibly a monoclonal antibody, is used which has been raised against such a synthetic peptide.
One peptide used in this disclosure is EKAHDGGR (SEQ ID NO:8). Generally, the peptides are chosen to include the cross-linking site K.
It was reported in Bonde et al., Journal of Bone and Mineral Research Vol. 10: S271--Abstract S481 (1995) that a polyclonal assay in which binding to antibodies is competed for between constituents of a sample and immobilized peptide of the sequence EKAHDGGR (SEQ ID NO:8) will respond to sample containing the hexapeptide AHDGGR (SEQ ID NO:9).
A poster exhibited by the authors at the ASBMR meeting in 1995 disclosed that this octapeptide was EKAH.beta.DGGR (SEQ ID NO:3) (i.e., the isomerized form).
WO96/30765 reports that some "peptide" fragments in body fluid have amino acid sequences that differ from each other by virtue of the isomerization of aspartic acid to isoaspartic acid (alternatively referred to as .beta.-aspartic acid). The term "peptide" is indicated here in quotes because such an isomerized aspartic acid residue does not form a peptide bond with the proximal C-terminal residue.
The isomerization has the effect of transferring that part of the peptide chain which runs downstreamn of the aspartic acid residue in the carboxyl terminus direction from the alpha carboxylic acid of the aspartic acid to which it is bonded via a peptide bond in the normal protein to the side chain carboxylic acid in a non-peptide amide bond, as shown below: ##STR1##
The isomerization of proteins containing aspartic acid has been reported previously to be a spontaneous reaction occurring under physiological conditions. See for instance Brennan et al., Protein Science 2: 331-338 (1993); Galletti et al., Biochem. J. 306: 313-325 (1995); Lowenson et al., Blood Cells 14: 103-117 (1988); and Oliya et al., Pharmaceutical Research, 11: 751 (1994).
Similar isomerization can occur in proteins containing asparagine residues (i.e., with --NH.sub.2 instead of --OH in the starting protein in the above reaction scheme).
The references described above focus on, or in some cases are limited to, antibody detection of peptides that are cross-linked, or at least contain the cross-linking residue or the cross-linkable site. None of these references report the detection or the effective use of peptides having no cross-linkable site.